ATP-dependent protease complexes degrade many unstable cellular proteins. These molecular machines function both generally in protein turnover, and specifically in the regulation of processes such as transcription, apoptosis, antigen presentation and cell cycle progression (Jesenberger et al. (2002) Nat. Rev. Mol. Cell Biol. 3:112). A high degree of conservation is evident among them; the archaebacterial and eukaryotic 20S proteolytic core particles share both sequence and structural homology (Bochtler et al. (1999) Ann. Rev. Biophys. Biomol. Struct. 28:295), while eubacteria have functionally related complexes: ClpYQ, ClpXP and ClpAP (Bochtler et al. (2000) Nature 403:800; Bochtler et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94:6070; Groll et al. (1997) Nature 386:463). The 20S core particle (CP) is composed of four stacked heptameric rings structured in an α-β-β-α configuration. Access to the proteolytic central chamber is obstructed at both ends of the cylindrical assembly by N-terminal projections of the α-subunits, thus preventing uncontrolled proteolytic degradation (Groll et al. (1997) Nature 386:463; Whitby et al. (2000) Nature 408:115; Groll et al. (2000) Nat. Struct. Biol. 7:1062). In eukaryotes, docking with the 19S regulatory particle (RP) to form the complete 26S proteasome is sufficient to relieve this block, opening a channel into the core (Groll et al. (2000) Nat. Struct. Biol. 7:1062; Kohler et al. (2001) Mol. Cell 7:1143).
Eukaryotes have evolved an elaborate system that operates in conjunction with the proteasome to facilitate the temporal and specific regulation of intracellular proteolysis. Substrates targeted for degradation by the proteasome pathway are recognized by the E1, E2, and E3 ubiquitin conjugation machinery and tagged with polyubiquitin chains, which are thought to promote the proteolytic process through their binding with the proteasome. These three consecutively acting enzymes are necessary for target recognition, transfer of a ubiquitin moiety to the substrate, and subsequent elongation of the ubiquitin branched chain (Hershko et al. (1998) Ann. Rev. Biochem. 67:425). Modularity and the large number of E2 Ub-conjugating enzymes and E3 Ub-ligases allow for great specificity and flexibility in detecting a diverse range of substrates. Once a protein is polyubiquitinated, it can be recognized and degraded by the 26S proteasome.
The polyubiquitin chain is thought to play two possible roles. The first is to target the protein to the proteasome and the second is to initiate the process of degradation. The targeting hypothesis is supported by the identification of several proteasome subunits that either bind or crosslink to ubiquitin chains (Deveraux et al. (1994) J. Biol. Chem. 269:7059; Lam et al. (2002) Nature 416:763). Hypotheses for how ubiquitin-dependent initiation of degradation might occur include: allosteric regulation, channel opening, and assistance in the unfolding of the target (Groll et al. (2003) Int. J. Biochem. Cell Biol. 35:606).